Therapeutic strategies in various discases include: nonspecific measures to mitigate or eliminate a cell dysfunction and prevent cell death; replacement of a missing or malfunctioning protein; introduction of functional nucleic acids (RNA or DNA) into cells to replace a mutated gene and introduction of novel genetic constructs to alter a cellular function. Advances in DNA technology have had a major impact on each of those therapeutic possibilities and nucleic acid transfer into diseased cells appears by far the most promising modality (Mulligan 1993, Science 260:926-932).
Viral vectors permit the expression of exogenous genes in eukaryotic cells, and thereby enable the production of proteins which require post-translational modifications unique to animal cells.
The wealth of information accumulated on adenoviruses over the last decades, has promoted them at the forefront of the gene therapy or immunization fields. Several features of adonoviruses make them attractive as gene transfer tools: (1) the structure of the adenoviral genome is well characterized; (2) large portions of viral DNA can be substituted by foreign sequences; (3) the recombinant variants are relatively stable, (4) the recombinant virus can be grown at high titer; (5) no human malignancy is associated with adenovirus; and (6) the use of attenuated wild-type adenovirus as a vaccine is safe.
Ad ate thus considered as very good vector candidates for in vivo gene transfer. Generally, such vectors are constructed by inserting the gene of interest in place of essential viral sequences such as E1 sequences (Berkner 1988 BioTechniques 6:616-629; Graham et al., 1991, Methods in Molecular Biology, 7:109-128, Ed: Murcy, The Human Press Inc.). This insertion results in an inactivation of the Ad since it can no longer replicate, hence the term replication-defective Ad. In order to propagates such vectors must be provided with the deleted element, (i.e. E1 proteins).
The elucidation of the nucleotide sequence of many Ad subtypes has enabled a precise characterization of the genomic organization thereof. The nucleotide sequence of human Ad5 is available from GenBank under accession number M73260. In simplistic terms adenoviruses comprise: (1) two inverted terminal repeats (ITR) at each end (5' and 3') which are essential for viral replication; (2) the early region 1 (E1) containing the E1A and E1B regions, both indispensible for replication, E1A and E1B are also required for complete transformation of various rodent cell lines, and polypeptide IX (pIX) which is essential For packaging of full-length viral DNA; and (3) the E2, E3 and E4 regions, with E3 being dispensable for replication (reviewed in Acsadi et al., 1995, J. Mol. Med. 73:165-180).
Recently, human Ad serotypes 2 and 5 have been used as vectors for efficient introduction of genes into several cell types both in vitro and in vivo (reviewed in Trapnell et al., 1994, Current Opinion Biotech. 5: 617-625; and Acsadi et al., 1995, J. Mol. Med. 73:165-180). Several factors need to be taken into consideration during the generation of Ad recombinants, among them is the impaired growth characteristics of some of them ( Imler et al., 1995 Gene Ther. 2:263-268; Massie et al., 1995 Bio/Technol. 13:602-608; and Schaack et al., 1995 J. Virol. 69: 3920-3923) which complicate the screening, propagation and production of high quality recombinant viral stocks with high titers (more than 10.sup.11 pfu/ml). Recently, critical issues relating to the characterization of such Ad vectors for gene therapy were reviewed in relation to clinical trials of the cystic fibrosis gene therapy (Engelhardt et al., 1993 Nature Genetics 4:27-34; Zabner et al., 1993 Cell 75:207-216; Boucher et al., 1994 Human Gene Ther. 5:615-639; Mittereder et al., 1994 Human gene Ther. 5:717-729; and Wilmot et al., 1996 Human Gene Ther. 7:301-318). Presently, a number of human clinical trials making use of Ad recombinants for the treatment of diseases like cystic fibrosis, Duchenne muscular dystrophy, and cancer, have started or are being considered (Lochmuller et al., 1994 Hum. Gene Ther. 5: 1485-1491). Potential sites for the insertion of a gene of interest in the recombinant Ad vectors comprise the E1 or E3 regions (i.e. E1+E3-deleted Ad recombinants) or the region between the end of the E4 and the beginning of the 3' ITR sequences. The majority of in vivo gene transfer experiments and human trials have been carried out using E1- and E3-deleted human type 2 or 5 adenoviruses. As alluded to above, E3-deleted recombinants are replication competent E1-deleted recombinants however, are unable to replicate and the missing E1 gene products are provide in trans by the E1-complementing cell line 293 (Lochmuller et al., 1994 Hum. Gene Ther. 5: 1485-1491). The 293 cells were established by stable transfection of a human embryonic kidney cell with adenoviral (human type 5) DNA containing the full length E1 region. The maximum deletion of up to 2.9 kb in the E1 region leaves intact the ITR sequence, the packaging signal at the left and of the adenoviral DNA (188-358 bp) and the pIX coding region (starting at 3507 bp). A useful E3 deletion was made by deletion of a 1.9 kb Xba I fragment (79 and 85 mu). These combined E1 and E3 deletions allowed for inserting approximately 7 kb of foreign DNA sequences in this first-generation recombinant. Extensions of the deletion in the E3 regions further increased the insert capacity to 8 kb, which meets the size requirements for most of the gene therapeutics (Bett et al., 1994 Proc. Natl. Acad. Sci. 91:8802-8806).
It is important to note that the recombinant Ad produced for clinical use have all been obtained using 293 cells (Graham et al., 1977, J. Gen. Virol. 36:59-72). Until the present invention, 293 cell line was the only available complementation cell line which efficiently expressed E1A and E1B RNAs and proteins. Unfortunately, it hats been documented that replication competent, also termed "revertant" virus can appear during multiple passages of the E1- and E3-deleted recombinant Ad in 293 cells, and eventually outgrow the original recombinant in large scale preparations (Lochmuller et al., 1994 Hum. Gene Ther. 5: 1485-1491). The E1 region is acquired from the 293 cells (and its derivatives) by homologous recombination at a very low frequency, but the E1-positive revertants seem to have a growth advantage with respect to their E1-negative counterparts. The presence of these revertants could thus jeopardize the safely of human gene therapy trials, especially when one considers the number of infectious viral particles required in certain applications. Experiments performed with mouse muscle have taught the use of of 2.times.10.sup.9 virus particulars to transduce more than 80% of the muscle fibers, since a human muscle is 2500 times larger, that would translate in the use of approximately 10.sup.12 -10.sup.13 viral particulars to inject only one human muscle. Supposing the presence of as little as 1/10 particules of E1+ revertants, in the stock, 10.sup.3 -10.sup.4 replication-competent particules would be injected in the muscle. It is clear that such an approach would fail to satisfy regulatory agencies.
Indeed, the 293 cells have been deemed "not suitable for large scale production of clinical grade material since batches are frequently contaminated with unacceptably high levels of replication competent adenovirus (RCA) arising through recombination" (Imler et al., 1996 Gene Ther. 3: 75-84). It should be stressed that the same authors have reported that numerous attempts to construct stable and efficient E1-complementing cell lines have failed and is therefore not a trivial task. In an attempt to solve this problem of RCA generation (Imler et al., 1996 Gene Ther. 3: 75-84) produced an E1-complementing cell line by stably transforming human lung A549 cells with E1 sequences containing the E1A, E1B and pIX regions. Novel A549 E1-complimenting cell lines were obtained which express high levels of E1 RNA and proteins. Strikingly however, the authors were unable to detect E1B protein expression in any of the A549 clones analyzed whether or not they produce high level of E1B RNA. Thus, the presence of a functional genetic unit does not necessarily predict that upon stable integration in the host, it will give rise to the expected proteins. It is also reported therein that the A549 clones, testing positive for infection with E1-deleted Ad vectors, showed a transformed phenotype and that the amplification yields therewith arc significantly lower than those obtained with 293 cells. Unfortunately, the generation of RCA with these A549 cells was not assessed. It should be noted that in the Imler et al., constructs, a significant overlap between the complementing element and the defective adenoviral vector occurs at the 3' end of the E1 region (approximately 700 bp). It follows that this overlap significantly increases the probabilities of homologous recombination and hence of the production of E1+ revertants. A disclosure of defective Ad vectors for the expression of exogenous nucleotide sequences in a host cell or organism, as well as vectors for the construction of E1-complementing cell lines, along the same lines is also found in the French publication to Imler et al., WO94/28152. However, this document fails to give an assessment of the yield of production of recombinant Ad by the complementation cell line, of the expression of the different adenoviral transcripts and proteins by the complementing cell line, and very importantly of the presence or absence of RCA during the production process leading to the obtention of the stock of defective Ad harboring the exogenous sequence of interest. It should be noted that WO94/28152 claims to diminish the problem of RCA production by deleting the 5' ITR (a non-substantiated declaration).
There still remains a need for the description of an E1-complementing cell line which combines at least one of the following properties: it expresses E1A and E1B proteins; it minimizes or abrogates the production of E1+ revertants; it is substantially as efficient as 293 or its derivatives in producing recombinant Ad; and it does not show a transformed and rounded phenotype. It would thus be advantageous to be provided with such E1-complementing cell lines which are efficient for the large scale production of E1-deleted Ad vectors devoid of RCA.
All of the above-cited citations are herein incorporated by reference.